The method for simultaneously detecting three viruses on tobacco by triple one-step RT-PCR is characterized in that: it is a method for simultaneously detecting tobacco common mosaic virus, potato virus Y and cucumber mosaic virus, and the method includes screening and comparison of conserved virus genomes Region, manual screening and synthesis of compound RT-PCR specific primers suitable for three viral diseases; analysis of TMV, PVY, CMV infected tobacco leaf samples; integration of enzymes and buffers required for reverse transcription and PCR reactions, through optimization of reaction conditions, To establish a method based on one-step RT-PCR combined detection of TMV, PVY and CMV infection in tobacco leaves. The triple one-step RT-PCR reaction adopted in the present invention has the advantages of simple and convenient operation, time-saving and labor-saving, reliable results, and low cost, and can effectively reduce the possibility of cross-contamination of samples during the operation process. , PVY, CMV monitoring and monitoring has a wide range of application prospects.